a) Field of the Invention
This invention relates to a method for the synthesis of certain novel analogs of parathyroid hormone and parathyroid hormone related peptide useful for the treatment of osteoporosis.
b) Description of Related Art
Osteoporosis is the most common form of metabolic bone disease and may be considered the symptomatic, fracture stage of bone loss (osteopenia). Although osteoporosis may occur secondary to a number of underlying diseases, 90% of all cases appear to be idiopathic. Postmenopausal women are particularly at risk for idiopathic osteoporosis (postmeno-pausal or Type I osteoporosis). Another high risk group for idiopathic osteoporosis is the elderly of either sex (senile or Type II osteoporosis). Osteoporosis has also been related to corticosteroid use, immobilization or extended bed rest, alcoholism, diabetes, gonadotoxic chemotherapy, hyperprolactinemia, anorexia nervosa, primary and secondary amenorrhea, and oophorectomy.
In the various forms of osteoporosis, bone fractures, which are the result of bone loss that has reached the point of mechanical failure, frequently occur. Postmenopausal osteoporosis is characterized by fractures of the wrist and spine, while femoral neck fractures seem to be the dominant feature of senile osteoporosis.
The mechanism by which bone is lost in osteoporotics is believed to involve an imbalance in the process by which the skeleton renews itself. This process has been termed bone remodeling. It occurs in a series of discrete pockets of activity. These pockets appear spontaneously within the bone matrix on a given bone surface as a site of bone resorption. Osteoclasts (bone dissolving or resorbing cells) are responsible for the resorption of a portion of bone of generally constant dimension. This resorption process is followed by the appearance of osteoblasts (bone forming cells) which then refill with new bone the cavity left by the osteoclasts.
In a healthy adult subject, the rate at which osteoclasts and osteoblasts are formed is such that bone formation and bone resorption are in balance. However, in osteoporotics an imbalance in the bone remodeling process develops which results in bone being lost at a rate faster than it is being made. Although this imbalance occurs to some extent in most individuals as they age, it is much more severe and occurs at a younger age in postmenopausal osteoporotics or following oophorectomy.
Adachi, et al. in Seminars in Arthritis and Rheumatism, 22:6, 375-84 (June 1993) report that despite much conflicting data regarding the pathophysiology of corticosteroid induced osteoporosis, it is generally agreed that there is a relative decrease in bone formation and a relative increase in bone resorption. Bone loss with resulting fractures and osteonecrosis is a frequent consequence of corticosteroid therapy. There is evidence that bone loss occurs rapidly within the first 6 to 12 months of corticosteroid therapy; there also appears to be a close relationship between rate of bone loss and corticosteroid dose. Men are equally susceptible to the effects of corticosteroids. The estimated incidence of fractures and osteonecrosis ranges from 30 to 50%.
There have been many attempts to treat osteoporosis with the goal of either slowing further bone loss or, more desirably, producing a net gain in bone mass. Certain agents, such as estrogen and the bisphosphonates, appear to slow further bone loss in osteoporotics. Agents which slow bone loss, because of the different durations of bone resorption and formation, may appear to increase bone mass (on the order of 3 to 7%). However, this apparent increase is limited in time, not progressive, and is due to a decrease in “remodeling space.” In addition, because of the close coupling between resorption and formation, treatments which impede bone resorption also ultimately impede bone formation.
It has been suggested that treatment with parathyroid hormone (PTH) would lead to both increased bone turnover and a positive calcium balance. However, human clinical trials have shown that any increase in trabecular bone is offset by a decrease in cortical bone, so that there is no net increase in total bone.
Hefti, et al. in Clinical Science 62, 389-396 (1982) have reported that daily subcutaneous doses of either bPTH(1-84) or hPTH(1-34) increased whole body calcium and ash weight of individual bones in both normal and osteoporotic adult female rats.
Liu, et al. in J. Bone Miner. Res. 6:10, 1071-1080 (1991) have noted that ovariectomy of adult female rats induced a 47% loss in the percentage of trabecular bone in the proximal tibial metaphysis, accompanied by a significant increase in the number of osteoblasts and trabecular osteoclasts. Daily subcutaneous injections of hPTH(1-34) completely reversed the loss of trabecular bone and resulted in amounts of trabecular bone exceeding that of sham operated controls. The number of osteoblasts increased and the number of osteoclasts decreased.
Hock et al. in J. Bone Min. Res. 7:1, 65-71 (1992) have reported that daily subcutaneous injections of hPTH(1-34) to healthy adult male rats for 12 days increased trabecular and cortical bone calcium and dry weight. Total bone mass, trabecular bone volume, trabecular thickness and number, and osteoblastic surfaces were increased.
The mammalian parathyroid hormones, e.g. human (hPTH), bovine (bPTH), and porcine (pPTH), are single polypeptide chains of 84 amino acid residues, with molecular weights of approximately 9500. Biological activity is associated with the N-terminal portion, with residues (1-34) apparently the minimum required.
The N-terminal segment of human PTH differs from the N-terminal segment of the bovine and porcine hormones by only three and two amino acid residues, respectively:
hPTH(1-34):Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu(SEQ ID NO:1)1                5                  10                  15Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp                20                  25                  30Val His Asn Phe;bPTH(1-34):Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys His Leu(SEQ ID NO:2)1                5                   10                 15Ser Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp                 20                  25                 30Val His Asn Phe;pPTH(1-34):Ser Val Ser Glu Ile G1n Leu Met His Asn Leu Gly Lys His Leu(SEQ ID NO:3)1                5                  10                  15Ser Ser Leu Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp                20                   25                 30Val His Asn Phe.
The primary function of PTH is to elicit the adaptive changes that serve to maintain a constant concentration of Ca2+ in the extracellular fluid. PTH acts on the kidneys to increase tubular reabsorption of Ca2+ from the urine, as well as stimulating the conversion of calcifediol to calcitriol, which is responsible for absorption of Ca2+ from the intestines. One prominent effect is to promote the mobilization of Ca2+ from bone. PTH acts on bone to increase the rate of resorption of Ca2+ and phosphate. PTH stimulates the rate of bone resorption by osteoclasts, increases the rate of differentiation of mesenchymal cells to osteoclasts, and prolongs the half life of these latter cells. With prolonged action of PTH the number of bone forming osteoblasts is also increased; thus, the rate of bone turnover and remodeling is enhanced. However, individual osteoblasts appear to be less active than normal.
Rosenblatt, et al. in U.S. Pat. Nos. 4,423,037, 4,968,669 and 5,001,223 have disclosed PTH antagonists obtained by the deletion of the N-terminal (1-6) amino acids and the selective replacement of Phe7, Met8,18, and Gly12. Tyr34-NH2 reportedly increased the activity and stability of these compounds.
Parathyroid hormone-related peptide (PTHrp), a 140+ amino acid protein, and fragments thereof, reproduce the major biological actions of PTH. PTHrp is elaborated by a number of human and animal tumors and other tissues and may play a role in hypercalcemia of malignancy. The sequence of hPTHrp (1-34) is as follows:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile(SEQ ID NO:4)1               5                   10                   15Gln Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu                20                  25                   30Ile His Thr Ala.
The sequence homology between hPTH and hPTHrp is largely limited to the 13 N-terminal residues, 8 of which are identical; only 1 of 10 amino acids in the (25-34) receptor binding region of hPTH is conserved in hPTHrp. Conformational similarity may underlie the common activity. Cohen, et al. in J. Biol. Chem. 266:3, 1997-2004 (1991) have suggested that much of the sequence of PTH(1-34) and PTHrp(1-34), in particular regions (5-18) and (21-34), assumes an α-helical configuration, while noting that there is some question whether this configuration prevails for the carboxyl terminal end under physiological conditions. Such a secondary structure may be important for lipid interaction, receptor interaction, and/or structural stabilization.
We have synthesized analogs of PTH and of PTHrp with the objective of developing improved therapeutic agents for the restoration of bone mass in mammalian subjects, including those afflicted with osteoporosis.
The compounds of this invention are disclosed in U.S. Pat. No. 5,589,452, filed Jul. 14, 1992, issued Dec. 31, 1996, and in pending U.S. patent application Ser. No. 08/184,328, filed Jan. 18, 1994, and published in International Publication No. WO 94/01460 on Jan. 20, 1994, filed as International Application No. PCT/US93/06465 on Jul. 13, 1993. The foregoing are incorporated by reference herein. We now report an improved method for the synthesis of these compounds.